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• Sleep enhances the migratory potential of T cells to lymph nodes in healthy humans. • Plasma from sleeping participants mimics the effects of sleep on T-cell migration. • Growth hormone and prolactin are the main hormonal mediators of this sleep effect. • These findings may explain adjuvant-like effects of sleep on vaccination responses. Sleep strongly supports the formation of adaptive immunity, e.g., after vaccination. However, the underlying mechanisms remain largely obscure. Here we show in healthy humans that sleep compared to nocturnal wakefulness specifically promotes the migration of various T-cell subsets towards the chemokine CCL19, which is essential for lymph-node homing and, thus, for the initiation and maintenance of adaptive immune responses. Migration towards the inflammatory chemokine CCL5 remained unaffected. Incubating the cells with plasma from sleeping participants likewise increased CCL19-directed migration, an effect that was dependent on growth hormone and prolactin signaling. These findings show that sleep selectively promotes the lymph node homing potential of T cells by increasing hormonal release, and thus reveal a causal mechanism underlying the supporting effect of sleep on adaptive immunity in humans. [ABSTRACT FROM AUTHOR]

Integrin LFA-1 plays a critical role in T-cell migration and in the formation of immunological synapses. LFA-1 functions through interacting with its ligands with differing affinities: low, intermediate, and high. Most prior research has studied how LFA-1 in the high-affinity state regulates the trafficking and functions of T cells. LFA-1 is also presented in the intermediate-affinity state on T cells, however, the signaling to activate LFA-1 to the intermediate-affinity state and the role of LFA-1 in this affinity state both remain largely elusive. This review briefly summarizes the activation and roles of LFA-1 with varied ligand-binding affinities in the regulation of T-cell migration and immunological synapse formation. [ABSTRACT FROM AUTHOR]

In pancreatic ductal adenocarcinoma (PDAC), the infiltration of CD8+ cytotoxic T cells (CTLs) is an important factor in determining prognosis. The migration pattern and interaction behavior of intratumoral CTLs are pivotal to tumor rejection. NLRP3-dependent proinflammatory cytokines IL-1β and IL-18 play a prominent role for CTL induction and differentiation. Here, we investigate the effects of T-cellular IL-1R and IL-18R signaling for intratumoral T-cell motility. Murine adenocarcinoma cell line Panc02 was stably transfected with ovalbumin (OVA) and fluorophore H2B-Cerulean to generate PancOVA H2B-Cerulean tumor cells. Dorsal skinfold chambers (DSFC) were installed on wild-type mice, and PancOVA H2B-Cerulean tumor cells were implanted into the chambers. PancOVA spheroids were formed using the Corning® Matrigel®-based 3D cell culture technique. CTLs were generated from OT-1 mice, Il1r−/− OT-1 mice, or Il18r−/− OT-1 mice and were marked with fluorophores. This was followed by the adoptive transfer of CTLs into tumor-bearing mice or the application into tumor spheroids. After visualization with multiphoton microscopy (MPM), Imaris software was used to perform T-cell tracking. Imaris analysis indicates a significantly higher accumulation of Il18r−/− CTLs in PancOVA tumors and a significant reduction in tumor volume compared to wild-type CTLs. Il18r−/− CTLs covered a longer distance (track displacement length) in comparison to wild-type (WT) CTLs, and had a higher average speed (mean track speed). The analysis of instantaneous velocity suggests a higher percentage of arrested tracks (arrests: <4 μm/min) for Il18r−/− CTLs. Our data indicate the contribution of IL-18R signaling to T-cell effector strength, warranting further investigation on phenomena such as intratumoral T-cell exhaustion. [ABSTRACT FROM AUTHOR]

Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1), two clinically relevant targets for the immunotherapy of cancer, are negative regulators of T-cell activation and migration. Optimizing the therapeutic response to CTLA-4 and PD-1 blockade calls for a more comprehensive insight into the coordinated function of these immune regulators. Mathematical modeling can be used to elucidate nonlinear tumor–immune interactions and highlight the underlying mechanisms to tackle the problem. Here, we investigated and statistically characterized the dynamics of T-cell migration as a measure of the functional response to these pathways. We used a previously developed three-dimensional organotypic culture of patient-derived tumor spheroids treated with anti-CTLA-4 and anti-PD-1 antibodies for this purpose. Experiment-based dynamical modeling revealed the delayed kinetics of PD-1 activation, which originates from the distinct characteristics of PD-1 and CTLA-4 regulation, and followed through with the modification of their contributions to immune modulation. The simulation results show good agreement with the tumor cell reduction and active immune cell count in each experiment. Our findings demonstrate that while PD-1 activation provokes a more exhaustive intracellular cascade within a mature tumor environment, the time-delayed kinetics of PD-1 activation outweighs its preeminence at the individual cell level and consequently confers a functional dominance to the CTLA-4 checkpoint. The proposed model explains the distinct immunostimulatory pattern of PD-1 and CTLA-4 blockade based on mechanisms involved in the regulation of their expression and may be useful for planning effective treatment schemes targeting PD-1 and CTLA-4 functions. [ABSTRACT FROM AUTHOR]

Integrin LFA-1 plays a critical role in T-cell migration and in the formation of immunological synapses. LFA-1 functions through interacting with its ligands with differing affinities: low, intermediate, and high. Most prior research has studied how LFA-1 in the high-affinity state regulates the trafficking and functions of T cells. LFA-1 is also presented in the intermediate-affinity state on T cells, however, the signaling to activate LFA-1 to the intermediate-affinity state and the role of LFA-1 in this affinity state both remain largely elusive. This review briefly summarizes the activation and roles of LFA-1 with varied ligand-binding affinities in the regulation of T-cell migration and immunological synapse formation.

In pancreatic ductal adenocarcinoma (PDAC), the infiltration of CD8+ cytotoxic T cells (CTLs) is an important factor in determining prognosis. The migration pattern and interaction behavior of intratumoral CTLs are pivotal to tumor rejection. NLRP3-dependent proinflammatory cytokines IL-1β and IL-18 play a prominent role for CTL induction and differentiation. Here, we investigate the effects of T-cellular IL-1R and IL-18R signaling for intratumoral T-cell motility. Murine adenocarcinoma cell line Panc02 was stably transfected with ovalbumin (OVA) and fluorophore H2B-Cerulean to generate PancOVA H2B-Cerulean tumor cells. Dorsal skinfold chambers (DSFC) were installed on wild-type mice, and PancOVA H2B-Cerulean tumor cells were implanted into the chambers. PancOVA spheroids were formed using the Corning® Matrigel®-based 3D cell culture technique. CTLs were generated from OT-1 mice, Il1r−/− OT-1 mice, or Il18r−/− OT-1 mice and were marked with fluorophores. This was followed by the adoptive transfer of CTLs into tumor-bearing mice or the application into tumor spheroids. After visualization with multiphoton microscopy (MPM), Imaris software was used to perform T-cell tracking. Imaris analysis indicates a significantly higher accumulation of Il18r−/− CTLs in PancOVA tumors and a significant reduction in tumor volume compared to wild-type CTLs. Il18r−/− CTLs covered a longer distance (track displacement length) in comparison to wild-type (WT) CTLs, and had a higher average speed (mean track speed). The analysis of instantaneous velocity suggests a higher percentage of arrested tracks (arrests: Il18r−/− CTLs. Our data indicate the contribution of IL-18R signaling to T-cell effector strength, warranting further investigation on phenomena such as intratumoral T-cell exhaustion.

Abstract Background The brain barriers establish compartments in the central nervous system (CNS) that significantly differ in their communication with the peripheral immune system. In this function they strictly control T-cell entry into the CNS. T cells can reach the CNS by either crossing the endothelial blood–brain barrier (BBB) or the epithelial blood-cerebrospinal fluid barrier (BCSFB) of the choroid plexus (ChP). Objective Analysis of the cellular and molecular mechanisms involved in the migration of different human CD4+ T-cell subsets across the BBB versus the BCSFB. Methods Human in vitro models of the BBB and BCSFB were employed to study the migration of circulating and CNS-entry experienced CD4+ T helper cell subsets (Th1, Th1*, Th2, Th17) across the BBB and BCSFB under inflammatory and non-inflammatory conditions in vitro. Results While under non-inflammatory conditions Th1* and Th1 cells preferentially crossed the BBB, under inflammatory conditions the migration rate of all Th subsets across the BBB was comparable. The migration of all Th subsets across the BCSFB from the same donor was 10- to 20-fold lower when compared to their migration across the BBB. Interestingly, Th17 cells preferentially crossed the BCSFB under both, non-inflamed and inflamed conditions. Barrier-crossing experienced Th cells sorted from CSF of MS patients showed migratory characteristics indistinguishable from those of circulating Th cells of healthy donors. All Th cell subsets could additionally cross the BCSFB from the CSF to ChP stroma side. T-cell migration across the BCSFB involved epithelial ICAM-1 irrespective of the direction of migration. Conclusions Our observations underscore that different Th subsets may use different anatomical routes to enter the CNS during immune surveillance versus neuroinflammation with the BCSFB establishing a tighter barrier for T-cell entry into the CNS compared to the BBB. In addition, CNS-entry experienced Th cell subsets isolated from the CSF of MS patients do not show an increased ability to cross the brain barriers when compared to circulating Th cell subsets from healthy donors underscoring the active role of the brain barriers in controlling T-cell entry into the CNS. Also we identify ICAM-1 to mediate T cell migration across the BCSFB.

The trafficking of T-cells through peripheral tissues and into afferent lymphatic vessels is essential for immune surveillance and an adaptive immune response. Glycogen synthase kinase 3β (GSK3β) is a serine/threonine kinase and regulates numerous cell/tissue-specific functions, including cell survival, metabolism, and differentiation. Here, we report a crucial involvement of GSK3β in T-cell motility. Inhibition of GSK3β by CHIR-99021 or siRNA-mediated knockdown augmented the migratory behavior of human T-lymphocytes stimulated via an engagement of the T-cell integrin LFA-1 with its ligand ICAM-1. Proteomics and protein network analysis revealed ongoing interactions among GSK3β, the surface receptor Notch1 and the cytoskeletal regulator CRMP2. LFA-1 stimulation in T-cells reduced Notch1-dependent GSK3β activity by inducing phosphorylation at Ser9 and its nuclear translocation accompanied by the cleaved Notch1 intracellular domain and decreased GSK3β-CRMP2 association. LFA-1-induced or pharmacologic inhibition of GSK3β in T-cells diminished CRMP2 phosphorylation at Thr514. Although substantial amounts of CRMP2 were localized to the microtubule-organizing center in resting T-cells, this colocalization of CRMP2 was lost following LFA-1 stimulation. Moreover, the migratory advantage conferred by GSK3β inhibition in T-cells by CHIR-99021 was lost when CRMP2 expression was knocked-down by siRNA-induced gene silencing. We therefore conclude that GSK3β controls T-cell motility through interactions with CRMP2 and Notch1, which has important implications in adaptive immunity, T-cell mediated diseases and LFA-1-targeted therapies.

Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1), two clinically relevant targets for the immunotherapy of cancer, are negative regulators of T-cell activation and migration. Optimizing the therapeutic response to CTLA-4 and PD-1 blockade calls for a more comprehensive insight into the coordinated function of these immune regulators. Mathematical modeling can be used to elucidate nonlinear tumor–immune interactions and highlight the underlying mechanisms to tackle the problem. Here, we investigated and statistically characterized the dynamics of T-cell migration as a measure of the functional response to these pathways. We used a previously developed three-dimensional organotypic culture of patient-derived tumor spheroids treated with anti-CTLA-4 and anti-PD-1 antibodies for this purpose. Experiment-based dynamical modeling revealed the delayed kinetics of PD-1 activation, which originates from the distinct characteristics of PD-1 and CTLA-4 regulation, and followed through with the modification of their contributions to immune modulation. The simulation results show good agreement with the tumor cell reduction and active immune cell count in each experiment. Our findings demonstrate that while PD-1 activation provokes a more exhaustive intracellular cascade within a mature tumor environment, the time-delayed kinetics of PD-1 activation outweighs its preeminence at the individual cell level and consequently confers a functional dominance to the CTLA-4 checkpoint. The proposed model explains the distinct immunostimulatory pattern of PD-1 and CTLA-4 blockade based on mechanisms involved in the regulation of their expression and may be useful for planning effective treatment schemes targeting PD-1 and CTLA-4 functions.

Human induced pluripotent stem cell (hiPSC)‐derived blood‐brain barrier (BBB) models established to date lack expression of key adhesion molecules involved in immune cell migration across the BBB in vivo. Here, we introduce the extended endothelial cell culture method (EECM), which differentiates hiPSC‐derived endothelial progenitor cells to brain microvascular endothelial cell (BMEC)‐like cells with good barrier properties and mature tight junctions. Importantly, EECM‐BMEC‐like cells exhibited constitutive cell surface expression of ICAM‐1, ICAM‐2, and E‐selectin. Pro‐inflammatory cytokine stimulation increased the cell surface expression of ICAM‐1 and induced cell surface expression of P‐selectin and VCAM‐1. Co‐culture of EECM‐BMEC‐like cells with hiPSC‐derived smooth muscle‐like cells or their conditioned medium further increased the induction of VCAM‐1. Functional expression of endothelial ICAM‐1 and VCAM‐1 was confirmed by T‐cell interaction with EECM‐BMEC‐like cells. Taken together, we introduce the first hiPSC‐derived BBB model that displays an adhesion molecule phenotype that is suitable for the study of immune cell interactions. [ABSTRACT FROM AUTHOR]

When migratory T cells encounter antigen‐presenting cells (APCs), they arrest and form radially symmetric, stable intercellular junctions termed immunological synapses which facilitate exchange of crucial biochemical information and are critical for T‐cell immunity. While the cellular processes underlying synapse formation have been well characterized, those that maintain the symmetry, and thereby the stability of the synapse, remain unknown. Here we identify an antigen‐triggered mechanism that actively promotes T‐cell synapse symmetry by generating cytoskeletal tension in the plane of the synapse through focal nucleation of actin via Wiskott–Aldrich syndrome protein (WASP), and contraction of the resultant actin filaments by myosin II. Following T‐cell activation, WASP is degraded, leading to cytoskeletal unraveling and tension decay, which result in synapse breaking. Thus, our study identifies and characterizes a mechanical program within otherwise highly motile T cells that sustains the symmetry and stability of the T cell–APC synaptic contact. Synopsis: When naïve T cells detect cognate antigens on the surface of antigen presenting cells, these highly migratory cells undergo immediate arrest and form radially symmetric cell‐cell conjugate interfaces termed immunological synapses. The stability of such synapses is a crucial determinant of T cell activation, but the T cell‐intrinsic mechanisms that regulate synaptic lifetime are not clear. This study finds that T cells construct specialized actin architectures within the immunological synapse to elevate cytoskeletal tension, thereby reinforcing synaptic stability. In T cells, lamellar protrusions would constantly attempt to break the synapse. Integrin activation is insufficient to sustain the synapse.Antigen recognition‐induced actin foci and associated actomyosin arrangements generate high in‐plane cytoskeletal tension that actively restrains synapse breaking.Via nucleation of actin foci, WASP acts as a central regulator of this mechanism. Downregulation of WASP following T cell activation leads to loss of foci‐dependent actin architecture resulting into synapse unravelling.T cells that lack WASP are predisposed to synapse symmetry breaking, irrespective of the substrate stiffness. [ABSTRACT FROM AUTHOR]

Background: The brain barriers establish compartments in the central nervous system (CNS) that significantly differ in their communication with the peripheral immune system. In this function they strictly control T-cell entry into the CNS. T cells can reach the CNS by either crossing the endothelial blood–brain barrier (BBB) or the epithelial blood-cerebrospinal fluid barrier (BCSFB) of the choroid plexus (ChP). Objective: Analysis of the cellular and molecular mechanisms involved in the migration of different human CD4+ T-cell subsets across the BBB versus the BCSFB. Methods: Human in vitro models of the BBB and BCSFB were employed to study the migration of circulating and CNS-entry experienced CD4+ T helper cell subsets (Th1, Th1*, Th2, Th17) across the BBB and BCSFB under inflammatory and non-inflammatory conditions in vitro. Results: While under non-inflammatory conditions Th1* and Th1 cells preferentially crossed the BBB, under inflammatory conditions the migration rate of all Th subsets across the BBB was comparable. The migration of all Th subsets across the BCSFB from the same donor was 10- to 20-fold lower when compared to their migration across the BBB. Interestingly, Th17 cells preferentially crossed the BCSFB under both, non-inflamed and inflamed conditions. Barrier-crossing experienced Th cells sorted from CSF of MS patients showed migratory characteristics indistinguishable from those of circulating Th cells of healthy donors. All Th cell subsets could additionally cross the BCSFB from the CSF to ChP stroma side. T-cell migration across the BCSFB involved epithelial ICAM-1 irrespective of the direction of migration. Conclusions: Our observations underscore that different Th subsets may use different anatomical routes to enter the CNS during immune surveillance versus neuroinflammation with the BCSFB establishing a tighter barrier for T-cell entry into the CNS compared to the BBB. In addition, CNS-entry experienced Th cell subsets isolated from the CSF of MS patients do not show an increased ability to cross the brain barriers when compared to circulating Th cell subsets from healthy donors underscoring the active role of the brain barriers in controlling T-cell entry into the CNS. Also we identify ICAM-1 to mediate T cell migration across the BCSFB. [ABSTRACT FROM AUTHOR]

Here we report on the development of a breakthrough microfluidic human in vitro cerebrovascular barrier (CVB) model featuring stem cell-derived brain-like endothelial cells (BLECs) and nanoporous silicon nitride (NPN) membranes (µSiM-CVB). The nanoscale thinness of NPN membranes combined with their high permeability and optical transparency makes them an ideal scaffold for the assembly of an in vitro microfluidic model of the blood–brain barrier (BBB) featuring cellular elements of the neurovascular unit (NVU). Dual-chamber devices divided by NPN membranes yield tight barrier properties in BLECs and allow an abluminal pericyte-co-culture to be replaced with pericyte-conditioned media. With the benefit of physiological flow and superior imaging quality, the µSiM-CVB platform captures each phase of the multi-step T-cell migration across the BBB in live cell imaging. The small volume of <100 µL of the µSiM-CVB will enable in vitro investigations of rare patient-derived immune cells with the human BBB. The µSiM-CVB is a breakthrough in vitro human BBB model to enable live and high-quality imaging of human immune cell interactions with the BBB under physiological flow. We expect it to become a valuable new tool for the study of cerebrovascular pathologies ranging from neuroinflammation to metastatic cancer. [ABSTRACT FROM AUTHOR]

Centrosome- and Golgi-localized protein kinase N-associated protein (CG-NAP), also known as AKAP450, is a cytosolic scaffolding protein involved in the targeted positioning of multiple signaling molecules, which are critical for cellular functioning. Here, we show that CG-NAP is predominantly expressed in human primary T-lymphocytes, localizes in close proximity (

T-cell migration across the blood-brain barrier (BBB) is a crucial step in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple scle rosis (MS). Two-photon intravital microscopy (2P-IVM) has been established as a powerful tool to study cell-cell interactions in inflammatory EAE lesions in living animals. In EAE, central nervous system inflammation is strongly pronounced in the spinal cord, an organ in which 2P-IVM imaging is technically very challenging and has been limited to the lumbar spinal cord. Here, we describe a novel spinal cord window preparation allowing to use 2P-IVM to image immune cell interactions with the cervical spinal cord microvascular endothelium during EAE. We describe differences in the angioarchitecture of the cervical spinal cord versus the lumbar spinal cord, which will entail different hemodynamic parameters in these different vascular beds. Using T cells as an example, we demonstrate the suitability of this novel methodology in imaging the post-arrest multistep T-cell extravasation across the cervical spinal cord microvessels. The novel methodology includes an outlook to the analysis of the cellular pathway of T-cell diapedesis across the BBB by establishing visualization of endothelial junctions in this vascular bed. [ABSTRACT FROM AUTHOR]

Treatment with soluble myelin peptide can efficiently and specifically induce tolerance to demyelination autoimmune diseases including multiple sclerosis, however the mechanism underlying this therapeutic effect remains to be elucidated. In actively induced mouse model of experimental autoimmune encephalomyelitis (EAE) we analyzed T cell and innate immune cell responses in the central nervous system (CNS) and spleen after intraperitoneal (i.p.) infusion of myelin oligodendrocyte glycoprotein (MOG). We found that i.p. MOG infusion blocked effector T cell recruitment to the CNS and protected mice from EAE and lymphoid organ atrophy. Innate immune CD11b + cells preferentially recruited MOG-specific effector T cells, particularly when activated to become competent antigen presenting cells (APCs). During EAE development, mature APCs were enriched in the CNS rather than in the spleen, attracting effector T cells to the CNS. Increased myelin antigen exposure induced CNS-APC maturation, recruiting additional effector T cells to the CNS, causing symptoms of disease. MOG triggered functional maturation of splenic APCs. MOG presenting APCs interacted with MOG-specific T cells in the spleen, aggregating to cluster around CD11b + cells, and were trapped in the periphery. This process was MHC II dependent as an MHC II directed antibody blocked CD4 + T cell cluster formation. These findings highlight the role of myelin peptide-loaded APCs in myelin peptide-induced EAE and immune tolerance. [ABSTRACT FROM AUTHOR]

Viral clearance requires effector T-cell egress from the draining lymph node (dLN). The mechanisms that regulate the complex process of effector T-cell egress from the dLN after infection are poorly understood. Here, we visualized endogenous pathogen-specific effector T-cell migration within, and from, the dLN. We used an inducible mouse model with a temporally disrupted sphingosine-1-phosphate receptor-1 (S1PR1) gene specifically in endogenous effector T cells. Early after infection, WT and S1PR1-/- effector T cells localized exclusively within the paracortex. This localization in the paracortex by CD8 T cells was followed by intranodal migration by both WT and S1PR1-/- T cells to positions adjacent to both cortical and medullary lymphatic sinuses where the T cells exhibited intense probing behavior. However, in contrast to WT, S1PR1-/- effector T cells failed to enter the sinuses. We demonstrate that, even when LN retention signals such as CC chemokine receptor 7 (CCR7) are down-regulated, T cell intrinsic S1PR1 is the master regulator of effector T-cell emigration from the dLN. [ABSTRACT FROM AUTHOR]

Intrathymic T-cell differentiation is a key process for the development and maintenance of cell-mediated immunity, and occurs concomitantly to highly regulated migratory events. We have proposed a multivectorial model for describing intrathymic thymocyte migration. One of the individual vectors comprises interactions mediated by laminins (LMs), a heterotrimeric protein family of the extracellular matrix. Several LMs are expressed in the thymus, being produced by microenvironmental cells, particularly thymic epithelial cells (TECs). Also, thymocytes and epithelial cells express integrin-type LM receptors. Functionally, it has been reported that the dy/dy mutant mouse (lacking the LM isoform 211) exhibits defective thymocyte differentiation. Several data show haptotactic effects of LMs upon thymocytes, as well as their adhesion on TECs; both effects being prevented by anti-LM or anti-LM receptor antibodies. Interestingly, LM synergizes with chemokines to enhance thymocyte migration, whereas classe-3 semaphorins and B ephrins, which exhibit chemorepulsive effects in the thymus, downregulate LM-mediated migratory responses of thymocytes. More recently, we showed that knocking down the ITGA6 gene (which encodes the a6 integrin chain of LM receptors) in human TECs modulates a large number of cell migration-related genes and results in changes of adhesion pattern of thymocytes onto the thymic epithelium. Overall, LM-mediated interactions can be placed at the cross-road of the multivectorial process of thymocyte migration, with a direct influence per se, as well as by modulating other molecular interactions associated with the intrathymic-trafficking events. [ABSTRACT FROM AUTHOR]

The integrin lymphocyte function associated antigen-1 (LFA-1) plays a key role in leukocyte trafficking and in adaptive immune responses through interactions with adhesive ligands, such as ICAM-1. Specific blockade of these interactions has validated LFA-1 as a therapeutic target in many chronic inflammatory diseases, however LFA-1 antagonists have not been clinically successful due to the development of a general immunosuppression, causing fatal side effects. Growing evidence has now established that LFA-1 mediates an array of intracellular signaling pathways by triggering a number of downstream molecules. In this context, a class of multimodular domain-containing proteins capable of recruiting two or more effector molecules, collectively known as "adaptor proteins," has emerged as important mediators in LFA-1 signal transduction. Here, we provide an overview of the adaptor proteins involved in the intracellular signaling cascades by which LFA-1 regulates T-cell motility and immune responses. The complexity of the LFA-1-associated signaling delineated in this review suggests that it may be an important and challenging focus for future research, enabling the identification of "tunable" targets for the development of immunotherapies. [ABSTRACT FROM AUTHOR]

Rho-GTPases belong to the Ras superfamily and are crucial signal transducing proteins downstream of many receptors. In general, the Rho-GTPases function as molecular switches, cycling between inactive (GDP-bound) and active (GTP-bound) states. The activated GTP bound Rho-GTPases interact with a broad spectrum of effectors to regulate a plethora of biological pathways including cytoskeletal dynamics, motility, cytokinesis, cell growth, apoptosis, transcriptional activity and nuclear signaling. Recently, gene targeting in mice allowed the selective inactivation of different Rho-GTPases and has advanced our understanding of the physiological role of these proteins, particularly in the immune system. Particularly, these proteins are key signaling molecules in T lymphocytes, which are generated in the thymus and are major players in the immune system. The scope of this review is to discuss recent data obtained in Rho-GTPases deficient mice by focusing on the role-played by Rho-GTPases in T-lymphocyte development, migration, activation and differentiation. [ABSTRACT FROM PUBLISHER]

Fibroblastic reticular cells (FRCs), through their expression of CC chemokine ligand (CCL)19 and CCL21, attract and retain T cells in lymph nodes (LNs), but whether this function applies to both resting and activated T cells has not been examined. Here we describe a model for conditionally depleting FRCs from LNs based on their expression of the diphtheria toxin receptor (DTR) directed by the gene encoding fibroblast activation protein-α (FAP). As expected, depleting FAP+ FRCs causes the loss of naive T cells, B cells, and dendritic cells from LNs, and this loss decreases the magnitude of the B- and T-cell responses to a subsequent infection with influenza A virus. In contrast, depleting FAP+ FRCs during an ongoing influenza infection does not diminish the number or continued response of activated T and B cells in the draining LNs, despite still resulting in the loss of naïve T cells. Therefore, different rules govern the LN trafficking of resting and activated T cells; once a T cell is engaged in antigen-specific clonal expansion, its retention no longer depends on FRCs or their chemokines, CCL19 and CCL21. Our findings suggest that activated T cells remain in the LN because they down-regulate the expression of the sphingosine-1 phosphate receptor-1, which mediates the exit of lymphocytes from secondary lymphoid organs. Therefore, LN retention of naive lymphocytes and the initiation of an immune response depend on FRCs, but is an FRC independent and possibly cell-autonomous response of activated T cells, which allows the magnitude of clonal expansion to determine LN egress. [ABSTRACT FROM AUTHOR]

Background & Aims: The enterohepatic circuit of T cells may be responsible for the development of autoimmune liver disease. We employed transgenic mice to characterize phenotype and migration patterns of CD8 T cells activated in liver and gut. Methods: We studied the migration of antigen-specific CD8 T cells primed in liver or gut after transfer in wild-type mice or mice that express ovalbumin in liver or gut. We performed transcriptome analysis of these two distinct T cell populations and confirmed our findings by flow cytometry. Results: Specific migration patterns were induced by activation of CD8 T cells in gut or liver. Gut-activated CD8 T cells expressed α4β7 and CCR9 and migrated to the gut and to the liver. Liver-activated T cells expressed integrins α4, α6, β1, α4β7 as well as CD62L, Ly6C, and neuropilin-1 and retained the capability to re-circulate through lymph nodes. Presence of the antigen increased retention of both types of activated T cells in the liver, but migration of liver-activated T cells to the gut was prohibited. Conclusions: CD8 T cells primed in the liver in vivo are not capable of migrating to the gut, implying that the enterohepatic circuit of CD8 T cells is in fact a one-way road from the gut to the liver. Priming of CD8 T cells in the liver results in a distinct phenotype with attributes of central memory cells and induces a unique homing pattern. Gut-primed T cells preferentially home to the liver, in principle enabling them to induce autoimmune liver disease. [Copyright &y& Elsevier]

Tissues such as the genital tract, skin, and lung act as barriers against invading pathogens. To protect the host, incoming microbes must be quickly and efficiently controlled by the immune system at the portal of entry. Memory is a hallmark of the adaptive immune system, which confers long-term protection and is the basis for efficacious vaccines. While the majority of existing vaccines rely on circulating antibody for protection, struggles to develop antibody-based vaccines against infections such as herpes simplex virus ( HSV) and human immunodeficiency virus ( HIV) have underscored the need to generate memory T cells for robust antiviral control. The circulating memory T-cell population is generally divided into two subsets: effector memory (TEM) and central memory (TCM). These two subsets can be distinguished by their localization, as TCM home to secondary lymphoid organs and TEM circulate through non-lymphoid tissues. More recently, studies have identified a third subset, called tissue-resident memory (TRM) cells, based on its migratory properties. This subset is found in peripheral tissues that require expression of specific chemoattractants and homing receptors for T-cell recruitment and retention, including barrier sites such as the skin and genital tract. In this review, we categorize different tissues in the body based on patterns of memory T-cell migration and tissue residency. This review also describes the rules for TRM generation and the properties that distinguish them from circulating TEM and TCM cells. Finally, based on the failure of recent T-cell-based vaccines to provide optimal protection, we also discuss the potential role of TRM cells in vaccine design against microbes that invade through the peripheral tissues and highlight new vaccination strategies that take advantage of this newly described memory T-cell subset. [ABSTRACT FROM AUTHOR]

We describe a new approach for interactive analysis of time-lapse microscopy, and apply this approach to elucidating whether polarity regulation is conserved between epithelial cells and lymphocytes. A key advantage of our analysis platform, 'TACTICS', is the capacity to visualize individual data points in the context of large data sets, similar to standard approaches in flow cytometry. Scatter plots representing microscopic parameters or their derivations such as polarity ratios are linked to the original data such that clicking on each dot enables a link to images and movies of the corresponding cell. Similar to flow cytometric analysis, subsets of the data can be gated and reanalyzed to explore the relationships between different parameters. TACTICS was used to dissect the regulation of polarization of the cell fate determinant, Numb, in migrating lymphocytes. We show here that residues of Numb that are phosphorylated by atypical protein kinase C (aPKC) to mediate apicobasal polarity in epithelial cells are not required for polarization of Numb in T cells, indicating that the role of aPKC is not conserved between lymphocytes and epithelia. [ABSTRACT FROM AUTHOR]

In an accompanying paper, we find specific localization of diabetogenic T cells only to islets of Langerhans bearing the specific antigen. Instrumental in the specific localization was the presence of intraislet dendritic cells bearing the β-cell-peptide-MHC complex. Here, we report that the entry of diabetogenic CD4 T cells very rapidly triggered inflammatory gene expression changes in islets and vessels by up-regulating chemokines and adhesion molecules. Vascular cell adhesion molecule-1 (VCAM-1) expression was notable in blood vessels, as was intercellular adhesion molecule-1 (ICAM-1). ICAM-1 was also found on β-cells. These expression changes induced the entry of nonspecific T cells that otherwise did not localize to the islets. In contrast to the entry of diabetogenic CD4 T cells, the entrance of nonspecific T cells required a chemokine response and VCAM-1 expression by the islets. IFN-γ was important for the early gene expression changes in the islets. By microarray analysis, we detected up-regulation of a group of IFN-inducible genes as early as 8 h post-T-cell transfer. These studies establish that entry of diabetogenic T cells induces a state of receptivity of islets to subsequent immunological insults. [ABSTRACT FROM AUTHOR]

Understanding the entry of autoreactive T cells to their target organ is important in autoimmunity because this entry initiates the inflammatory process. Here, the events that lead to specific localization of diabetogenic CD4 T cells into islets of Langerhans resulting in diabetes were examined. This was evaluated in two models, one in which T cells specific for a hen-egg white lysozyme (HEL) peptide were injected into mice expressing HEL on β cells and the other using T cells in the nonobese diabetic mouse strain, which develops spontaneous diabetes. Only T cells specific for β-cell antigens localized in islets within the first hours after their injection and were found adherent to intraislet dendritic cells (DCs). DCs surrounded blood vessels with dendrites reaching into the vessels. Localization of antigen-specific T cells did not require chemokine receptor signaling but involved class II histocompatibility and intercellular adhesion molecule 1 molecules. We found no evidence for nonspecific localization of CD4 T cells into normal noninflamed islets. Thus, the anatomy of the islet of Langerhans permits the specific localization of diabetogenic T cells at a time when there is no inflammation in the islets. [ABSTRACT FROM AUTHOR]

The mucosal immune system is constantly exposed to a wide range of commensal and potentially pathogenic microbial species. Chronic exposure to foreign organisms makes generation of an appropriate immune response critical in maintaining a balance between elimination of harmful pathogens, peaceful coexistence with commensals, and prevention of autoimmunity. Intestinal intraepithelial lymphocytes provide a first line of defense at this extensive barrier with the outside world, and as such, understanding their role in immunity is critical. [ABSTRACT FROM AUTHOR]

Abstract: Immune adaptors SLP-76, ADAP and SKAP1 (SKAP-55) play central roles in anti-CD3 induced ‘inside-out’ signalling for LFA-1 activation and ICAM-1 adhesion. However, it has been unclear whether SKAP1 is also required for chemokine-induced T-cell motility. In this study, we found that SDF-1 and CCL21 induced similar motility in SKAP1 deficient (SKAP1−/−) and wild type (SKAP1+/+) resting, primary T-cells. In addition, the speed (i.e. 13μM/min), tracking distance (i.e. length) and displacement values (i.e. direct distance between the start and the end positions of cell movement) in response to SDF1 were similar for SKAP1−/− and SKAP1+/+ primary, activated T-cells. Relatively high strength anti-CD3 ligation also arrested the migration (i.e. stop-signal) of resting SKAP1+/+ and SKAP1−/− T-cells in the presence of SDF-1 and CCL21. These data demonstrate that contrary to its central role in anti-CD3 induced LFA-1 adhesion, the response of primary T-cells to SDF-1 and CCL21 is not profoundly dependent on SKAP1 expression. [Copyright &y& Elsevier]

The ordered, directional migration of T-lymphocytes is a key process during immune surveillance, immune response, and development. A novel series of pyrrolo-1,5-benzoxazepines have been shown to potently induce apoptosis in variety of human chemotherapy resistant cancer cell lines, indicating their potential in the treatment of both solid tumors and tumors derived from the hemopoietic system. Pyrrolobenzoxazepine 4-acetoxy-5-(1-naphtyl)naphtho[2,3- b]pyrrolo[1,2- d][1,4]-oxazepine (PBOX-15) has been shown to depolymerize tubulin in vitro and in the MCF7 breast cancer cell line. We hypothesized that this may suggest a role for this compound in modulating integrin-induced T-cell migration, which is largely dependent on the microtubule dynamics. Experiments were performed using human T lymphoma cell line Hut78 and peripheral blood T-lymphocytes isolated from healthy donors. We observed that human T-lymphocytes exposed to PBOX-15 have severely impaired ability to polarize and migrate in response to the triggering stimulus generated via cross-linking of integrin lymphocyte function associated antigen-1 receptor. Here, we show that PBOX-15 can dramatically impair microtubule network via destabilization of tubulin resulting in complete loss of the motile phenotype of T-cells. We demonstrate that PBOX-15 inhibitory mechanisms involve decreased tubulin polymerization and its post-translational modifications. Novel microtubule-targeting effects of PBOX-15 can possibly open new horizons in the treatment of overactive inflammatory conditions as well as cancer and cancer metastatic spreading. [ABSTRACT FROM AUTHOR]

The trafficking of T-cells through peripheral tissues and into afferent lymphatic vessels is essential for immune surveillance and an adaptive immune response. Glycogen synthase kinase 3β (GSK3β) is a serine/threonine kinase and regulates numerous cell/tissue-specific functions, including cell survival, metabolism, and differentiation. Here, we report a crucial involvement of GSK3β in T-cell motility. Inhibition of GSK3β by CHIR-99021 or siRNA-mediated knockdown augmented the migratory behavior of human T-lymphocytes stimulated via an engagement of the T-cell integrin LFA-1 with its ligand ICAM-1. Proteomics and protein network analysis revealed ongoing interactions among GSK3β, the surface receptor Notch1 and the cytoskeletal regulator CRMP2. LFA-1 stimulation in T-cells reduced Notch1-dependent GSK3β activity by inducing phosphorylation at Ser9 and its nuclear translocation accompanied by the cleaved Notch1 intracellular domain and decreased GSK3β-CRMP2 association. LFA-1-induced or pharmacologic inhibition of GSK3β in T-cells diminished CRMP2 phosphorylation at Thr514. Although substantial amounts of CRMP2 were localized to the microtubule-organizing center in resting T-cells, this colocalization of CRMP2 was lost following LFA-1 stimulation. Moreover, the migratory advantage conferred by GSK3β inhibition in T-cells by CHIR-99021 was lost when CRMP2 expression was knocked-down by siRNA-induced gene silencing. We therefore conclude that GSK3β controls T-cell motility through interactions with CRMP2 and Notch1, which has important implications in adaptive immunity, T-cell mediated diseases and LFA-1-targeted therapies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Fazil, Prasannan, Wong, Kottaiswamy, Salim, Sze and Verma.)

We are interested in the characterization of the effects of alcohol on human T-cell activation, maturation, and migration, because this cell population is crucial in the initiation, regulation, and propagation of cellular immunity. We and others have described the effects of both acute and chronic exposure of human immune cells to ethanol (EtOH) in vitro. Herein, we briefly, review these reports and expand this body of literature with the inclusion of new data recently obtained in our laboratory. We confirm the blunting effects of EtOH on the production of interleukin-2 and mitogen proliferative response following T-cell mitogen stimulation, and on the expression of membrane markers of activation. We show that EtOH significantly alters the expression of the CD4 cell-associated marker of activation, CD26. We report the effect of EtOH on the expression of the homing receptor CD62L by CD4+ cells, and on their ability to adhere by a CD18-mediated process to a defined cellular substratum. Furthermore, we demonstrate the effects of EtOH and EtOH and 0-endor-phin pretreatment on the activation of CD4+ lymphocytes endowed with the homing receptor CD62L. [ABSTRACT FROM AUTHOR]

Regulatory T cells (Tregs) interfere with antitumor immune responses, hence facilitating oncogenesis and tumor progression. Our recent findings indicate that neuropilin 1, which is highly expressed by Tregs, mediates the infiltration of these cells into the tumor in a vascular endothelial growth factor (VEGF)-dependent manner, thereby contributing to cancer-associated immunosuppression. [ABSTRACT FROM AUTHOR]

Effector T-cells rely on integrins to drive adhesion and migration to facilitate their immune function. The heterodimeric transmembrane integrin LFA-1 (αLβ2 integrin) regulates adhesion and migration of effector T-cells through linkage of the extracellular matrix with the intracellular actin treadmill machinery. Here, we quantified the velocity and direction of F-actin flow in migrating T-cells alongside single-molecule localisation of transmembrane and intracellular LFA-1. Results showed that actin retrograde flow positively correlated and immobile actin negatively correlated with T-cell velocity. Plasma membrane-localised LFA-1 forms unique nano-clustering patterns in the leading edge, compared to the mid-focal zone, of migrating T-cells. Deleting the cytosolic phosphatase PTPN22, loss-of-function mutations of which have been linked to autoimmune disease, increased T-cell velocity, and leading-edge co-clustering of pY397 FAK, pY416 Src family kinases and LFA-1. These data suggest that differential nanoclustering patterns of LFA-1 in migrating T-cells may instruct intracellular signalling. Our data presents a paradigm where T-cells modulate the nanoscale organisation of adhesion and signalling molecules to fine tune their migration speed, with implications for the regulation of immune and inflammatory responses.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

The exploration screening of phenotypic changes in motile T-cells within a signaling environment has always been an arduous task due to the sheer population of these microscopic cells. In recent years, High-Content Analysis (HCA) has gained epochal momentum and has allowed for a wider range of quantitative multiplexed cell-based assays in the field of lymphocyte signaling. In this chapter, we consolidate our understanding and describe the technical approach and methodology to quantify T-cell migratory phenotypes using HCA. Optimizations to be adopted to generate high-quality cytological images of motile T-cells and subsequent analysis using HCA are detailed as well.

The immune system and its components defend our body against diverse pathogens and help in maintaining tissue homeostasis. Immune cells are highly dynamic in terms of their growth, migration, differentiation, and effector functions, and adopt diverse metabolic configurations to respond to varying immunological challenges. Growing body of evidence suggests that metabolic pathways fuel immune cells for their functioning, including T-cell migration to the site of infection. This chapter provides detailed methodology for the efficient extraction of T-cell metabolites for successful downstream immunometabolomic profiling of motile T-lymphocytes.

Regulated migration of T-lymphocytes through high endothelial venules and secondary lymphoid organs is necessary for an adaptive immune response. Uncontrolled trafficking of T-cells is implicated in many pathological conditions, including autoimmune disorders, such as psoriasis and inflammatory bowel disease. T-cell migration is regulated mainly by the αLβ2 integrin receptor LFA-1, which interacts primarily with its cognate ligand ICAM-1 expressed on the endothelium. This interaction triggers a plethora of downstream signaling pathways, which are not fully understood. Thus, in order to dissect the signal transduction processes at molecular levels and phenotypic changes in migrating T-cells, a laboratory model mimicking T-cell motility is important. Here, we describe a simple and highly reproducible in vitro model to study T-cell migration.

T-lymphocytes are the principle coordinators of the immune defense system and play a major role in the protection of our body against infections, intruders of non-self, and malignancies. To mount an immune response, T-cells need to be effectively employed to tissue sites of infection or inflammation and establish contacts with antigen-presenting cells (APCs) or malignant cells. Understanding how T-cells navigate toward their recruitment sites would offer new therapeutic opportunities. Advancement in the hardware and software upgrades of microscopy technology has created several efficient and easy-to-operate live cell imaging platforms. In this protocol, we present a generalized and simple-to-follow protocol for live cell imaging of migrating T-cells, which can also be adopted to visualize real-time tracking of intracellular signaling events.

The cycles of internalization of the cell surface β2 integrin receptor lymphocyte function-associated antigen 1 (LFA-1) and its re-exposure on the plasma membrane are important for T-cell trafficking. Biotinylation of cells enables to measure surface expression of receptors, and after reducing surface biotin with reducing buffer, enables to measure the internalization of receptors. Here, by using biotin in combination with reducing buffer and recombinant intercellular adhesion molecule-1 (rICAM-1)-coated dishes and subsequent Western immunoblot analysis, we describe how to measure internalization of the LFA-1 receptor and its re-expression back to the cell surface in motile T-lymphocytes.